Method for analysis through layer-by-layer sample removal using a cantilever probe

ABSTRACT

A method and apparatus for analysis of a sample. The method includes an accessing operation for accessing a region of the sample via a tip of at least one probe mounted on a cantilever. A removing operation removes a sample material from the region that is accessed by the tip of the at least one probe mounted on the cantilever. A sensing operation senses a parameter associated to the removal of the sample material in the removing operation. The accessing, removing, and sensing operations are repeated to facilitate removal of at least one layer of the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to EuropeanPatent Application No. 05111775.2 filed Dec. 7, 2005, the entire text ofwhich is specifically incorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention relates to a method for analysis of a sample. Moreparticularly, the present invention relates to a method forthree-dimensional analysis of a sample. The present invention alsoextends to an analyzer for analysis of a sample.

With the advent of scanning probe techniques such as the scanningtunneling microscope STM and the atomic force microscope AFM, it hasbecome possible to image a wide variety of samples with a resolutioncapability on the molecular or atomic scale.

An AFM image of a sample may be collected by mapping the deflection of acantilever-mounted probe as a function of the x-y position of the tip ofthe probe on the sample when the tip comes into contact with, or is inclose proximity to, the sample whilst being scanned relative thereto.This yields a topographic image of the sample surface. However, shouldthe sample have an undercut region or steep wall, only the height changeand not the profile thereof is accessible by the tip so that the imageobtained will not depict the true shape of the sample.

The aforementioned problem has been addressed in an article byWickramasinghe et. al in Applied Physics Letters, Volume 64(19),published 9 May 1994, pages 2498 to 2500, titled, “Method for imagingsidewalls by atomic force microscopy”. A two-dimensional (2D) AFM isdescribed in this article specifically designed to image samples withvertical profiles. This design involves: (i) the use of a modified tip,specifically, a cylindrical tip fabricated so as to have lowerprotrusions giving it a “boot” shape; (ii) vibration of the tip in thevertical Z direction and horizontal X direction, (iii) a two-dimensionalscan and servo system, and (iv) a mode of acquisition of data pointsthat is different from what is used for conventional AFM imaging.

The method proposed by Wickramasinghe et. al may be applicable toimaging biological samples such as cells, which, not only have verticaledges, typically, for example, at cell boundaries, but also haveirregular surfaces. Even so, this method would only yield thepossibility of performing a two-dimensional analysis of the profile ofsuch a sample.

Accordingly, it is desirable to provide a method capable of performing athree-dimensional analysis of a sample, particularly a biologicalsample, which has the atomic/molecular resolution capability of AFMwhilst retaining simplicity of design, i.e. not requiring the use ofspecialized tips, servo- and scanning systems, for example.

BRIEF SUMMARY OF THE INVENTION

According to an embodiment according to a first aspect of the presentinvention, there is provided a method for analysis of a sample includingthe steps of: (i) accessing a region of the sample via a tip of at leastone probe mounted on a cantilever; (ii) removing a sample material fromthe region that is accessed by the tip of the at least one probe mountedon the cantilever; and (iii) sensing a parameter associated to theremoval of the sample material in step (ii), wherein steps (i) to (iii)are repeated to facilitate removal of at least one layer of the sample.

In a step (i), a tip of a cantilever-mounted probe is brought intocontact with a surface of a sample of interest so that the tip accessesa region of the sample. In a step (ii), the sample material is removedfrom the region where the tip has accessed the sample. In a step (iii),removal of the sample material is sensed using at least one sensor. Asensing signal produced by the sensor provides information on, forexample, the character and/or strength with which the sample material isremoved. Steps (i) to (iii) are repeated for the tip being scannedacross the sample so that a whole layer of the sample is removed. Bycollecting the sensing signal produced by the sensor at each scan pointof the tip on the sample, a map of the profile of the sample can beobtained.

According to an embodiment of the first aspect, steps (i) to (iii) arerepeated to facilitate removal of at least another layer of the sample.

The above-described process is repeated for the removal of subsequentlayers of the sample. Combining the measurements performed above for allthe layers of the sample that are removed yields a three-dimensionalimage of the sample. The possibility of obtaining three-dimensionalinformation is advantageous in certain fields, especially in thebiological field where three-dimensional information of biologicalsystems may be used in, for example, drug discovery and to provide aninsight into the dynamics of such systems.

According to an embodiment of the first aspect, the sample material thatis removed in step (ii) from the region that is accessed by the tip ofthe at least one probe mounted on the cantilever is one of an atom ofthe sample and a molecule of the sample.

The tip of the cantilever-mounted probe can, for example, be of the typeused for conventional AFM imaging. Such a tip can be used for accessinga localized region of the sample, such as, for example, an atom ormolecule of the sample, which is then treated to facilitate the removalof sample material from that region.

According to an embodiment of the first aspect, there is furtherprovided a step of removing debris from a surface of the sample, thedebris resulting from the removal of the sample material in step (ii)from the region that is accessed by the tip of the at least one probemounted on the cantilever.

It could be that removal of the sample material in step (ii) results insome debris on the surface thereof. In order that the debris does notinterfere with, for example, scanning of the tip to other points on thesample and/or sensing of a parameter associated to the removal of thesample material, it is desirable to remove the debris. Any appropriatedevice and/or method may be used for removing the debris off the samplesurface. An example of a device that may be used is aspecially-fabricated miniature fan.

According to an embodiment of the first aspect, in step (iii), sensingof the parameter associated to the removal of the sample material isselected to be done by an integrated sensor that is integrated into thecantilever onto which the at least one probe is mounted. In this case,the integrated sensor may comprise one of: a vibration sensor, a chargesensor, a temperature sensor and a combination of a vibration sensor, acharge sensor and a temperature sensor.

Sensing of the parameter associated to the removal of the samplematerial in step (iii) can, for example, be done by measuring aparameter of the cantilever onto which the probe is mounted and/or aparameter of the sample. Where a parameter of the cantilever is sensed,the corresponding sensor may be integrated into the cantilever for easeof implementation. In this case, the integrated sensor may be avibration sensor to detect a change in vibration of the cantilever inresponse to the removal of the sample material, a charge sensor todetect a change in a charge, for example, a surface charge, of thecantilever in response to the removal of the sample material, atemperature sensor in order to detect a change in temperature of thecantilever in response to the removal of the sample material or acombination of a vibration sensor, charge sensor and a temperaturesensor. An advantage associated to the use of the combination is thatthe sensing signal of the sensor, which has a higher magnitude out ofall the sensing signals that are produced by the sensors in thecombination, may be used. A further advantage is that, if one of thesensors is not functioning, then the other sensors in the combinationmay be used.

According to an embodiment of the first aspect, in step (iii), sensingof the parameter associated to the removal of the sample material isselected to be done by an external sensor. In this case, the externalsensor comprises one of: a pressure sensor, a smell sensor, amass-spectrometer and a combination of a pressure sensor, a smell sensorand a mass-spectrometer.

As discussed above, sensing of the parameter associated to the removalof the sample material in step (iii) can be done by measuring aparameter of the cantilever onto which the probe is mounted and/or aparameter of the sample. Measuring a parameter of the sample can be donevia an external sensor, which could be a smell sensor for sensing thesmell associated to the gaseous phase of the sample once it is removedby, for example, heating, a mass-spectrometer, a pressure sensor foroperation in a vacuum or a combination of a pressure sensor, a smellsensor and a mass-spectrometer. The advantages associated to the use ofthe combination correspond to those stated for the use of a combinationof sensors for the integrated sensor.

According to an embodiment of the first aspect, in step (iii), sensingof the parameter associated to the removal of the sample material isselected to be done by measuring a deflection of the cantilever ontowhich at the least one probe is mounted.

The removal of the sample material in step (ii) may be monitored bymeasuring a mechanical response of the cantilever, for example, via adeflection of the cantilever. The cantilever deflection can, forexample, be measured by directing a laser beam on its surface disposedopposite to that on which the probe is mounted and monitoring thedisplacement of the reflected laser beam on a quadrature-segmentedphotodiode.

According to an embodiment of the first aspect, removal of the samplematerial in step (ii) from the region that is accessed by the tip of theat least one probe mounted on the cantilever is selected to be done byheat applied to the tip of the at least one probe mounted on thecantilever. The heat applied to the tip of the at least one probemounted on the cantilever may be selected to be done by an integratedheater that is integrated into the cantilever onto which the at leastone probe is mounted. In this case, in step (iii), sensing of theparameter associated to the removal of the sample material may be doneby measuring the temperature of the integrated heater.

In step (ii), removal of the sample material from the region that isaccessed by the tip can be done heating the tip. The sample material is,in this case, “burnt off” or fragmented into a gaseous phase due to theenergy transfer from the hot tip. For ease of implementation, heat canbe applied to the tip via a heater that is integrated into thecantilever onto which the probe associated to that tip is mounted. Inorder to supplement the measurements obtained from the aforementionedinternal sensor and external sensor, removal of the sample material canbe sensed by the change in temperature this causes in the integratedheater.

According to an embodiment of the first aspect, the at least one layerof the sample is removed via a predetermined number of steps, the heatapplied to the tip of the at least one probe mounted on the cantileverbeing increased at each step relative to the previous step until thepredetermined number of steps are completed. In this case, a springconstant value of the cantilever onto which the at least one probe ismounted is switched to another spring constant value before the at leastanother layer of the sample is removed.

Where removal of the sample material is effected by application of heatvia the tip of the cantilever-mounted probe, the heat being applied tothe tip, for example, via the integrated heater, the removal of a layerof the sample material may be done via a predetermined number of steps.These steps may be defined by the number of scan points for scanning thetip relative to the sample. In this case, the heat applied to the tipcould be configured to increase with each scan point as it may be thatsome regions of the sample may require more heat than others for removalthereof. Before removal of the next layer in the above-described manner,a spring constant of the cantilever may be switched to another value. Bysimultaneously repeating the above-described heat treatment and, forexample, measuring the cantilever deflection at each scan point for eachof the spring constant values of the cantilever, additional informationon the removal process of the sample material can be obtained.

According to an embodiment of the first aspect, in step (iii), sensingof the parameter associated to the removal of the sample material isselected to be done by a combination of the integrated sensor, theexternal sensor, measuring the deflection of the cantilever onto whichthe at least one probe is mounted and measuring the temperature of theintegrated heater

By using a combination of the integrated sensor, the external sensor,measuring the deflection of the cantilever onto which the at least oneprobe is mounted and measuring the temperature of the integrated heaterto sense a parameter associated to the removal of the sample material,the sensitivity of the measurements can be increased. Furthermore, ifone of the sensors malfunctions, the other sensors in the combinationcan be relied upon.

According to an embodiment of the first aspect, the sample is abiological sample. In this case, the biological sample is frozen priorto step (i) being performed.

As discussed earlier, the possibility of obtaining three-dimensionalinformation is advantageous in certain fields, especially in thebiological field where three-dimensional information of biologicalsystems may be used in, for example, drug discovery and to provide aninsight into the dynamics of such systems. Here, a three-dimensionalanalysis of a biological system such as, for example, a cell may beperformed with molecular or atomic resolution. The possibility ofperforming the three-dimensional analysis with the resolution capabilityof, for example, an AFM is considered to outweigh the fact that a cellthat is being so analyzed will be destroyed due to cellular materialbeing destroyed at each point of contact of by the tip. Where the sampleto be analyzed is a biological sample, it is preferably frozen prior toperforming the analysis for ease of accessing a region on it by the tipand also so that a specialized servo-system is not required, which wouldbe the case if the sample were imaged without such pre-treatment and/orin a liquid, which is the medium of choice for imaging biologicalsamples. The biological sample can be frozen using known techniques, forexample, cryogenic techniques.

According to an embodiment of the first aspect, the shape of the tip ofthe at least one probe mounted on the cantilever is one of a conicalshape and a wedge shape.

The use of differently-shaped tips for performing the analysis of thesample has corresponding advantages. An advantage associated to aconically-shaped tip is ease of access to undercut regions of thesample. An advantage associated to a wedge-shaped tip is the possibilityto use it to remove a layer of the sample by “slicing” the layer.

According to an embodiment of a second aspect of the present invention,there is provided an analyzer for analysis of a sample comprising: a tipof at least one probe mounted on a cantilever to access a region of thesample; a sample remover configured to remove a sample material from theregion that is accessed by the tip of the at least one probe mounted onthe cantilever; and at least one sensor configured to sense a parameterassociated to the removal of the sample material from the region of thesample accessed by the tip of the at least one probe mounted on thecantilever, the analyzer being operable to remove at least one layer ofthe sample.

An embodiment of the second aspect benefits from all the advantages ofthe first aspect.

According to an embodiment of the second aspect, comprising an array ofprobes mounted on the cantilever.

Features of one aspect of the present invention may be applied to anyanother aspect and vice versa.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Reference will now be made, by way of example, to the accompanyingdrawings in which:

FIG. 1 illustrates the analyzer.

Within the description, the same reference numerals and/or signs areused to denote the same parts or the like.

DETAILED DESCRIPTION OF THE INVENTION

In the following, a description will be provided of the presentinvention through an embodiment of the present invention. However, thefollowing embodiments do not restrict the invention in the scope of theinvention and all combinations of features explained in the embodimentare not always essential to means of the invention for solving theproblems.

As can be seen from FIG. 1, a sample 1 to be analyzed is mounted onto asubstrate 2. A positioner 6 is used to position a tip 3 of at least oneprobe 4 mounted on a cantilever 5 relative to the sample 1.Specifically, the positioner 6 is used to bring the tip 3 into contactwith a surface of the sample 1. In this way, the tip 3 accesses a regionon the surface of the sample 1. A sample remover 11 is configured toremove a sample material from the region that is accessed by the tip 3.The sample remover 11 may be integrated into the cantilever 5 onto whichthe at least one probe 4 is mounted and/or may be provided as anexternal unit therefrom. At least one sensor 9, 10 is also provided inorder to sense a parameter associated to the removal of the samplematerial as facilitated by the sample remover 11. The at least onesensor may be an integrated sensor 9 being integrated into thecantilever 5 or it could be an external sensor 10 being providedseparately from the cantilever 5. A sensing signal S1 generated by theintegrated sensor 9 and/or a sensing signal S2 generated by the externalsensor 10 is conveyed to a processing unit 8, which processes thesesignals thereby to generate data that is transmitted to a datarepresentation unit 12 where the data may be further manipulated and/orgraphically represented. The processing unit 8 may be further configuredto convey the sensing signal S1 generated by the integrated sensor 9and/or the sensing signal S2 generated by the external sensor 10 to acontroller 7, which uses these signals for controlling the positioner 6and, by this, controlling the extent of contact between the tip 3 andthe sample 1.

The positioner 6 is operated so as to scan the tip 3 of the at least oneprobe 4 mounted on the cantilever 5 relative to the sample 1. The sampleremover 11 is operated to facilitate removal of the sample material ateach scan point. The internal sensor 9 and/or external sensor 10 areoperated to sense a parameter associated to the removal of the samplematerial at each such scan point and to generate a corresponding sensingsignal S1, S2. In this way, the analyzer is operated to remove a wholelayer of the sample 1. The process is then repeated for removal ofsubsequent layers of the sample 1. Mapping either or both of the sensingsignals S1, S2 as a function of each scan point of the tip 3 on thesample 1 until a whole layer of the sample 1 is removed, repeating thisfor each subsequent layer that is removed and then combining the resultsobtained for all the layers of the sample 1 that are removed may be usedto generate a three-dimensional image of the sample 1. Processing andmanipulation of such results can be done in the processing unit 8 andgraphically represented on the data representation unit 12. In thiscase, the processing unit 8 and data representation unit 12 may beincorporated in a computer and processing and manipulation of theresults as described above may be done by running a computer simulationprogram on the computer.

The tip 3 of the at least one probe 4 mounted on the cantilever 5 can,for example, be of the type used for conventional AFM imaging. Such atip can be used for accessing a localized region of the sample, such as,for example, an atom or molecule of the sample, which is then treatedvia the sample remover 11 to facilitate the removal of the atom ormolecule from that region. By removing a layer of the sample 1atom-by-atom or molecule-by-molecule in the above-described manner,repeating this for the removal of subsequent layers of the sample 1 andthen combining the results obtained for all the removed layers yields athree-dimensional image of the sample 1 would provide information on thesample 1 on an atomic or molecular scale.

Of course, different shapes for the tip 3 may be used in the analyzerfor performing the analysis of the sample 1 with each different shape ofthe tip 3 having an associated advantage. For example, aconically-shaped tip 3 may provide ease of access to undercut regions ofthe sample 1. An advantage associated to a wedge-shaped tip 3 is thepossibility to use it to remove a layer of the sample 1 by “slicing” thelayer.

It could be that removal of the sample material at each point of contactbetween the tip 3 and the sample 1, when the tip 3 is scanned relativeto the sample 1, results in some debris on the surface thereof. In orderthat the debris does not interfere with, for example, scanning of thetip 3 to other points on the sample 1 and/or disrupting operation of theat least one sensor 9, 10 in sensing a parameter associated to theremoval of the sample material, the debris is removed. Any appropriatedevice and/or method may be used in conjunction with the analyzer forremoving the debris off the sample surface, for example, aspecially-fabricated miniature fan can be used.

As mentioned earlier, the at least one sensor is an integrated sensor 9that is integrated into the cantilever 5 or an external sensor 10. Theintegrated sensor 9 and/or the external sensor 10 senses a parameterassociated to the removal of the sample material via the sample remover11. Typically, the integrated sensor 9 performs this task by measuring aparameter of the cantilever 5 into which it is integrated. Theintegrated sensor 9 may be a vibration sensor to detect a change invibration of the cantilever 9 in response to the removal of the samplematerial, a charge sensor to detect a change in a charge, for example, asurface charge, of the cantilever 9 in response to the removal of thesample material, a temperature sensor in order to detect a change intemperature of the cantilever 9 in response to the removal of the samplematerial or a combination of a vibration sensor, charge sensor and atemperature sensor. An advantage associated to the use of thecombination is that the sensing signal of the sensor, which has a highermagnitude out of all the sensing signals that are produced by thesensors in the combination, may be used. A further advantage is that, ifone of the sensors is not functioning, then the other sensors in thecombination may be used. Of course, the analyzer is not limited to theuse of the aforementioned sensors for the integrated sensor 9. Any othersensors may be used either independently or in combination to achievethe function of the integrated sensor 9.

Typically, the external sensor 10 senses a parameter of the sample 1 tosense the removal of the sample material. The external sensor 10 couldbe a smell sensor for sensing the smell associated to the gaseous phaseof the sample once it is removed by, for example, heating, amass-spectrometer, a pressure sensor for when the analysis is conductedin a vacuum or a combination of a pressure sensor, a smell sensor and amass-spectrometer. The advantages associated to the use of thecombination correspond to those stated for the use of a combination ofsensors for the integrated sensor. Of course, the analyzer is notlimited to the use of the aforementioned sensors for the external sensor10. Any other sensors may be used either independently or in combinationto achieve the function of the external sensor 10.

An alternative to using the integrated sensor 9 and/or the externalsensor 10 for sensing the parameter associated to the removal of thesample material is to measure a mechanical response of the cantilever 5to the removal of the sample material. Specifically, a deflection of thecantilever 5 can be measured. In this case, the deflection of thecantilever 5 can be measured by directing a laser beam on its surfacedisposed opposite to that on which the probe 4 is mounted and monitoringthe displacement of the reflected laser beam on, for example, aquadrature-segmented photodiode. The difference in a magnitude of thelaser beam as displaced on at least two of the segments of thequadrature-segmented photodiode may be used to measure the deflection ofthe cantilever 5.

Removal of the sample material from the region of the sample that isaccessed by the tip 3 can be done heating the tip. The sample materialis, in this case, “burnt off” or fragmented into a gaseous phase due tothe energy transfer from the hot tip. Removal of the sample materialcan, in this case, be facilitated by the sample remover 11 being aheater that is integrated into the cantilever 5 onto which the probe 4associated to that tip 3 is mounted. In order to supplement themeasurements obtained from the aforementioned internal sensor 9 andexternal sensor 10, a parameter of the sample remover 11 can be measuredin response to the removal of the sample material. In this case, theparameter that is measured is the change in temperature of theintegrated heater in response to the removal of the sample material.

Where removal of the sample material is effected by application of heatvia the tip 3, the heat being applied to the tip 3, for example, via thesample remover 11 being an integrated heater, the removal of a layer ofthe sample material may be done via a predetermined number of steps.These steps may be defined by the number of scan points for scanning thetip 3 relative to the sample 1. In this case, the heat applied to thetip 3 could be configured to increase with each scan point as it may bethat some regions of the sample 1 may require more heat than others forremoval thereof. Before removal of the next layer in the above-describedmanner, a spring constant of the cantilever 5 may be switched to anothervalue. By simultaneously repeating the above-described heat treatmentand, for example, measuring the deflection of the cantilever 5 at eachscan point for each of the spring constant values of the cantilever 5,additional information on the removal process of the sample material canbe obtained.

Although the removal of the sample material as facilitated by the sampleremover 11 has been described above in relation to the independent useof the integrated sensor 9, the external sensor 10, measuring thedeflection of the cantilever 5 onto which the at least one probe 4 ismounted and measuring the temperature of the integrated heater 11, theycan be used in combination for this purpose. An advantage associated tothe combination is that the sensitivity of the measurements can beincreased. Also, if one of the sensors malfunctions, the other sensorsin the combination can be relied upon. Furthermore, if, for example, thecantilever 5 has a spring constant value that makes it stiff and,therefore, insensitive, than the other sensors in the combination can beused.

The analyzer can be applied for three-dimensional analysis of biologicalsamples. In this case, the biological sample 1 would preferably befrozen prior to performing the analysis for ease of accessing a regionon it by the tip 3 since biological samples are known to have irregularsurfaces and also so that a specialized servo-system is not required,which would be the case if the sample 1 were imaged without suchpre-treatment and/or in a liquid, which is the medium of choice forimaging biological samples. Known cryogenic techniques may, for example,be used to freeze the biological sample 1.

Of course, the analyzer is not restricted to obtaining three-dimensionalinformation of only biological samples. For example, the sample 1 may bea metal or metal-containing substrate. In this case, the sample remover11 would preferably be configured to apply an electric field to the tip3 of the at least one probe 4 mounted on the cantilever 5 so as toremove material at the point of access on such a sample 1 by the tip 3.

Although the analyzer has been described with reference to the use of atleast one probe 4 mounted on a cantilever 5, a cantilever 5 with anarray of probes 4 mounted thereon can also be used.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated.

Having thus described the invention of the present application in detailand by reference to embodiments thereof, it will be apparent thatmodifications and variations are possible without departing from thescope of the invention defined in the appended claims.

1. A method for analysis of a sample comprising: accessing a region ofthe sample via a tip of at least one probe mounted on a cantilever,wherein the shape of the tip of is one of a conical shape and a wedgeshape; removing a sample material from the region that is accessed bythe tip of the at least one probe mounted on the cantilever, wherein thesample material is one of an atom of the sample and a molecule of thesample, wherein removal of the sample material is selected to beperformed by heat applied to the tip of the at least one probe mountedon the cantilever, and wherein the heat applied to the tip of the atleast one probe mounted on the cantilever is selected to be performed byan integrated heater that is integrated into the cantilever onto whichthe at least one probe is mounted; sensing a parameter associated to theremoval of the sample material in the removing operation, whereinsensing of the parameter associated to the removal of the samplematerial is selected to be done by a combination of an integrated sensorthat is integrated into the cantilever onto which the at least one probeis mounted, an external sensor, measuring a deflection of the cantileveronto which the at least one probe is mounted and measuring thetemperature of the integrated heater, wherein the integrated sensorcomprises one of: a vibration sensor, a charge sensor, a temperaturesensor and a combination of a vibration sensor, a charge sensor and atemperature sensor, and wherein the external sensor comprises one of: apressure sensor, a smell sensor, a mass-spectrometer and a combinationof a pressure sensor, a smell sensor and a mass-spectrometer; andremoving debris from a surface of the sample, the debris resulting fromthe removal of the sample material in the removing operation from theregion that is accessed by the tip of the at least one probe mounted onthe cantilever; and wherein the accessing, removing and sensingoperations are repeated to facilitate removal of at least one layer ofthe sample, wherein the at least one layer of the sample is removed viaa predetermined number of steps, the heat applied to the tip of the atleast one probe mounted on the cantilever being increased at each steprelative to the previous step until the predetermined number of stepsare completed, and wherein a spring constant value of the cantileveronto which the at least one probe is mounted is switched to anotherspring constant value before the at least another layer of the sample isremoved; wherein the accessing, removing and sensing operations arerepeated to facilitate removal of at least another layer of the sample;and wherein the sample is a biological sample which is frozen prior tothe accessing operation being performed.